ABSTRACT The effective repair of bone defects and fractures ? due to various etiologies such as trauma, iatrogenesis, congenital defects, or neoplasia ? remains a worldwide problem. The gold standard for repairing bone loss is the autograft. However, autografts are intrinsically limited in supply, difficult to fit at the injury site, and generate donor site morbidity. Since their clinical approval over 14 years ago, synthetic bone graft substitutes loaded with bone morphogenetic protein (BMP) were heralded as a promising alternative to conventional bone grafts. These substitutes are passive delivery systems loaded with supraphysiological levels of BMP (e.g., BMP2, BMP7); this has lead to significant morbidities such as painful, ectopic bone formation and life-threatening inflammatory reactions that are caused by off-target effects of the growth factor. In sharp contrast to the poorly controlled delivery of growth factor from conventional bone graft substitutes, expression of growth factors is tightly regulated, both spatially and temporally, during normal bone healing. Our long-term goal is to develop biomaterials that enable spatiotemporally-controlled delivery of regenerative factors in an externally-modulated, on-demand manner. The stimulus for such control is focused ultrasound (FUS), which is clinically-translatable since it can be applied non-invasively and delivered in a spatiotemporally controlled manner to sites deep within the body. The objective of this proposal is to develop an implantable scaffold where angiogenesis and osteogenesis, two critical processes in bone regeneration, are spatially- and temporally-controlled using FUS. The central hypothesis driving this project is that hyperthermia (i.e., 43-45oC), generated using FUS, can pattern blood vessel and bone growth by controlling the expression of angiogenic and osteogenic factors in cells containing heat shock-responsive gene switches. The switches utilized in this proposal have an additional level of control since they are also controlled by a ligand, thus yielding switches with exquisite specificity and low background expression in the unactivated state (i.e., in the absence of hyperthermia and ligand). The development of a potential breakthrough in bone regeneration creates a compelling rationale for the proposed research. The hypothesis will be tested via two specific aims: 1) Define acoustic parameters for FUS-dependent induction of transgene expression in vitro and in vivo; and 2) Demonstrate FUS-patterned blood vessel and bone growth in vivo following the induction of vascular endothelial growth factor (VEGF) or BMP2. In Aim 1, the acoustic properties of hydrogel scaffolds, which will contain the cells harboring the gene switch, will be optimized to maximize hyperthermic heating and cell viability. The scaffold composition and acoustic parameters identified in Aim 1 will be used in Aim 2 to generate blood vessels and bone in a mouse model. The proposed research is significant because it can lead to a new treatment for bone regeneration that is safer, more effective, and less invasive than existing therapeutic options. Overall, these gene switches could be broadly applied within regenerative medicine, including the development of two adjacent tissue types.